you may try, 4%PFA/ PBS fixation, 4C O/N.

Then, incubate the sample in 1:1 8% formic acid / 8% hydrochloric acid (~10 ml per sample should be far more than enough), 4C, 1 O/N.

After decalcification, rinse the sample with PBS for several times, make sure the acid are neutralized (some use ammonia solution in this step). 

Then, the sample should be ready for paraffin or OCT embedding.

 8% Hydrochloric Acid Stock Solution:

    Hydrochloric acid, concentrated ----------------- 40 ml

    Distilled water ------------------------------------- 460 ml

    Mix well and store at room temperature.

8% Formic Acid Stock Solution:

     Formic acid --------------------------------------- 40 ml

     Distilled water ------------------------------------ 460 ml

     Mix well and store at room temperature.

Hydrochloric Acid/Formic Acid Working Solution:

     Combine equal parts of the 8% hydrochloric acid solution and the 8% formic acid solution before use.

Ammonia Solution:

     Ammonia, Concentrated ------------------------ 5 drops

     Distilled water ----------------------------------- 100 ml

     Mix well

Dear Ka Kui Tong,

Thank you so much for your detail protocol.

Last week , after searching  protocol  in internet and taking suggestions from neighboring lab working on bone, I  did PFA fixation and left my samples in  5% formic acid . I checked the tissue status everyday. After 3- 4 overnights, it seems that the tissues( esp hindlimbs) are soft  and can be easily handled with lab scissors. The feet seems a little bit difficult to handle compared to hindlimb. The lab working on bone keep the samples for minimum 2 weeks with repeated change of formic acid change.

I was worried if prolonged decalcified it may interfere with lymph vessel related staining. The following day I will be embedding and staining. Hope everything goes well.

you can try this to test whether the tissue is fully decalcified

TESTING FOR DECALCIFICATION

Remove 5 ml of used decalcification fluid from the tissue processing vessel

Add ammonium hydroxide dropwise until the pH of the solution is neutral to pH paper.

 Add 5ml of saturated ammonium oxalate and shake well

 Allow to stand at room temperature for 30 minutes.

INTERPRETATION OF RESULTS

Formation of a precipitate (calcium hydroxide) after the addition of ammonium hydroxide indicates the presence of substantial amounts of calcium in the spent fluid.  The fluid should be changed and the tissue further decalcified.

Formation of a precipitate after ammonium oxalate addition shows less calcium.  If the solution remains clear for 30 minutes after addition of the oxalate, the tissue is essentially calcium free.

(ref. https://www.nationaldiagnostics.com/histology/article/decalcifying-tissue-histological-processing)

btw, the volume of the acid is critical. It should be at least 20 times of your sample tissues. otherwise, the acid solution will be saturated with the calcium and greatly reduce their decalcificatin ability. 

Also, overdecalcification can permanently damage a specimen. Especially some of the antigens.

We routinely de-calcify mouse paws by firstly fixing in formalin (24 -48 hours) then several changes of 10% EDTA pH 7.4 over three  weeks. You can check the extent of decalcification by X-ray. We have not observed any loss of integrity in morphology and  in IHC.

Thank you Ka Kui Tong,

I will take into account your suggestions when doing next time.

We also do de-calcification in 10% EDTA pH 7.4 for two weeks after 48h fixation in 4% buffered formalin. We don't have problems to stain vessels, muscle, etc. by IHC.

Thank you Richard and Melanie for adding information in this topic. I think i should try this as well.  Decalcification by EDTA sounds less harmful compared to formic acid without any loss of integrity of  sensitive samples. Definately time factor is longer with EDTA.

Thanx

Decalcification by chelating with EDTA is definitely the way to go when you want to do IHC on bone tissues. It is important that the tissue be well fixed beforehand.

We use this formula-

100g EDTA

16g Trizma Base

QS to 1L and pH to 7.4.

Tissue are suspended in the EDTA solution at 4C under constant agitation ( i.e. on a stir plate in the fridge). The EDTA solution is changed evry 4-5 days.

We check for complete decalcification by microCT, but you can aldo x-ray as others have mentioned

Yes, it takes longer than acid decalcification, but it is the best way to preserve antigenic epitopes. Good luck with your experiments! 

Osteosoft from Merck is our choice. Rapid and preserves the proteins well enough.

Thanx Ivan.